Vegetable fiber-digesting agent and method of processing vegetable waste by using the same

ABSTRACT

It is an object of the present invention to provide a process for decomposing vegetable waste materials easily, which solves problems such as the problem of treatment costs, the problem of energy, an environmental problem and the problem of time for treatment which are difficult to be solved by prior art.  
     The present invention provides an agent for decomposing plant fibrous materials, which contains plant fibrous materials decomposing enzymes derived from a microorganism, but not the microorganism. In addition, the present invention provides a process for treating vegetable waste materials comprising allowing the agent for decomposing plant fibrous materials to act on vegetable waste materials.

TECHNICAL FIELD

[0001] The present invention relates to an agent for decomposing plantfibrous materials and a process for treating vegetable waste materials.Specifically, the present invention relates to a process for treating ofvegetable waste materials such as rice straw and chaff by using plantfibrous materials decomposing enzymes obtained from aerobic bacteriaCellulomonas, anaerobic bacteria Clostridium and/or any othermicroorganisms which can produce plant fibrous materials decomposingenzymes.

BACKGROUND ART

[0002] As a process for treating waste materials comprising botanicaltissues generated during rice growing, a wheat production, production ofgrain such as corn and sugarcane, or production of legumes, adopted hasbeen a process wherein waste materials are incinerated in the fields; aprocess wherein waste materials are incinerated in an incinerator withfuel such as heavy oil or light oil; or a process wherein compost isprepared by mixing vegetable waste materials with sawdust, rice bran orthe like and piling the resultant up. However, the process wherein wastematerials are incinerated in an incinerator requires a large quantity offuel and the cost of transportation, and it has been considered whetherthe process wherein waste materials are incinerated in the fields shouldbe prohibited, because of a problem of environmental pollution caused bythe generated soot and smoke (a rice-straw pollution). Concerning theprocess of composting vegetable waste materials, adopted have been aprocess comprising mixing straw, sawdust, rice bran or the like tovegetable waste materials, sprinkling the mixture with slaked lime,piling the resulting mixture up out in the open and thereby letting itdecay naturally; and a process for preparing compost comprising mixingfinely-crushed vegetable waste materials with sawdust, chaff, chickendroppings or the like, regulating moisture of the mixture, addingfermentative bacteria, treated compost or the like to the resultant andstirring the resulting mixture in a container and whereby fermentationis carried out. However, according as agriculture is modernized andmechanized, the process for preparing compost tends not to be performedbecause of both labor and a period of time required to make the mixtureripe. Therefore, such waste materials tend to become industrial wastethough it is recognized as beneficial resources.

[0003] As a process other than those mentioned above, there is a processwherein vegetable waste materials are treated with microbiologicalpreparation. Vegetable waste materials are difficult to dissolve becauseof the structure, which is caused mainly by cellulose and/orhemicellulose in the vegetable waste materials. Because of this,cellulose and/or hemicellulose need to be hydrolyzed at least partly atthe first step so that the plant fibrous materials can be treated andthen used beneficially. As a process for hydrolyzing cellulose and/orhemicellulose, known is a process for treating wherein agents fordecomposing rice straw or the like comprising the mixture of many kindsof bacteria are used. In Japanese Unexamined Patent Publication No.157285/1996, described is a process for preparing liquid compost fromvegetable waste materials wherein at least one of pectin, cellulose andhemicellulose is decomposed with plant fibrous materials decomposingenzymes produced by fungi or bacteria. However, the process requiresthat a place for treatment should be obtained and provided withfacilities for the treatment, so that labor to transport vegetable wastematerials to the place is required. Furthermore, in Japanese UnexaminedPatent Publication No. 2613/1995, described is a bacterial preparationfor agriculture which is sprinkled directly to the fields. However, thedecomposition of vegetable waste materials by using the agent takes along time from sprinkling of the agent over the fields to observing theeffect, especially to the resulting vegetable waste materials becomingripe. The reason is that the vegetable waste materials are decomposedwith plant fibrous materials decomposing enzymes which microorganismsproduce after cultivation, i.e. that the decomposition of vegetablewaste materials requires the multiplication of the bacteria in soil.

[0004] For the purpose of allowing the existing bacterial preparationand lime-nitrogen agents to work effectively in cold districts such asTohoku and Hokkaido which are in a Japanese grain-growing district, itis insufficient to plow the agents in the fields once, so that it isnecessary to do that twice. At the time of rice growing, waste materialswhich are not decomposed ferment after rice planting to generate gas,and whereby taking root of young plant and growth of the roots areinhibited.

[0005] It is an object of the present invention to provide a process fordecomposing vegetable waste materials which solves problems such as theproblem of treatment costs mentioned above, the problem of energy, anenvironmental problem and the problem of time for treatment which aredifficult to be solved by prior art, without depending on livingconditions of microorganisms and without the need to transport wastematerials comprising plant fibrous materials.

DISCLOSURE OF INVENTION

[0006] The present invention provides an agent for decomposing plantfibrous materials which does not contain microorganisms but plantfibrous materials decomposing enzymes derived from microorganisms and aprocess for treating vegetable waste materials which comprises allowingthe agent for decomposing plant fibrous materials to act on vegetablewaste materials, and whereby the problems mentioned above can be solved.

[0007] An agent for decomposing plant fibrous materials of the inventioncan be obtained according to the embodiment of the invention describedas follows. In addition, a process for treating vegetable wastematerials of the present invention can be carried out according to thefollowing embodiment of the present invention. The present invention isexplained in detail hereinafter.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] The term “plant fibrous materials decomposing enzymes” as usedherein are enzymes derived from microorganisms which act on plantfibrous materials to decompose them in soil. Examples of plant fibrousmaterials decomposing enzymes are cellulase, xylase, pectinase,xylanase, pullulanase and glucosidase. The term “microorganism” as usedherein is not limited as long as the microorganism produces any plantfibrous materials decomposing enzymes, and a microorganism whichproduces at least cellulase and/or xylanase is preferred. Example ofmicroorganism is a microorganisms which belongs to a genus such asCellulomonas, Bacillus, Cellvibrio, Pseudomonas, Sporocytophaga,Acetivibrio, Clostridium, Bacterioides, Butyrivibrio, Treponema,Ruminococcus, Streptomyces, Thermoactinomyces, Thermonospola,Chaetomium, Humicola, Myceliophthola, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Acremonium, Agricus, Alternaria, Aspergillus,Botryodiplodia, Fusarium, Irpex, Myrothecium, Neurospora, Pellicularia,Penicillium, Pestalotiopsis, Pleurotus, Polyporus, Poria, pycnoporus,Pyricularia, Rhizopus, Schizophyllum, Sclerotium, Scytalidium,Termitomyces, Trametes or Trichoderma, and produces plant fibrousmaterials decomposing enzymes. Among such microorganisms, plant fibrousmaterials decomposing enzymes derived from a microorganism which belongsto Cellulomonas or Clostridium are preferred, and plant fibrousmaterials decomposing enzymes derived from Cellulomonas are morepreferred. Concrete examples of preferable species are Cellulomonas sp.K32A (National Institute of Bioscience and Human-Technology Agency ofIndustrial Science and Technology, accession number FERM BP-6766) strainand Clostridium cellulovorans ATCC 35296 strain. Because plant fibrousmaterials decomposing enzymes are allowed to work in the fields, theplant fibrous materials decomposing enzymes derived from mesophile arepreferred. The term “mesophile” used herein is a microorganism which cannot grow at both a high temperature range (at least 55° C.) and a lowtemperature range (at most 0° C.). Cellulase or xylanase activity can bemeasured by an usual method (Walfgang, H., Analitical Biochem., 164,72-77, 1987; Wood, T. M. et al., Methods of Enzymology, 160, 87-112,1988), so that a person skilled in the art can choose propermicroorganisms which produce plant fibrous materials decomposingenzymes. Furthermore, as plant fibrous materials decomposing enzymesused herein, included are enzymes obtained by preparing transformantsuch as the microorganisms or culture cells containing a plant fibrousmaterials decomposing enzyme gene derived from a microorganism on thebasis of gene-recombinant technique, and then cultivating thetransformant.

[0009] In plant fibrous materials decomposing enzymes, there areexo-type enzymes which decompose plant fibrous materials from endthereof and endo-type enzymes which decompose fibrous materials frominterior thereof. Both of them are preferred in the present invention.As a plant fibrous materials decomposing enzymes used in the presentinvention, preferred is an enzyme having the enzymatic activity at 10°C. to 60° C., preferably at 15° C. to 55° C. A person skilled in the artcan measure enzymatic activities by using a well known method. Forexample, enzymatic activity of plant fibrous materials decomposingenzymes such as cellulase or xylase, endoglucanase and xylanaseactivities can be measured by the usual method mentioned above(Walfgang, H., 1987, Analitical Biochem., 164, 72-77; Wood, T. M. etal., Methods of Enzymology, 160, 87-112, 1988).

[0010] The term “agent for decomposing plant fibrous materials” usedherein means an agent containing the plant fibrous materials decomposingenzymes described above, and an agent containing at least cellulaseand/or xylanase is preferred. The formulation of an agent fordecomposing plant fibrous materials is not particularly limited.Examples of formulation are liquid, powders and granules and the like.In addition, preferred is the agent for decomposing plant fibrousmaterials which contains both endo- and exo-types described above. Anagent for decomposing plant fibrous materials can be obtained bycultivating microorganisms which produce the plant fibrous materialsdecomposing enzymes described above. Cultivation of microorganisms isperformed by using proper medium under proper condition for eachmicroorganism. These medium and culture condition can be selected andset appropriately by a person skilled in the art. As a source of carbonfor medium, for example, used can be every kinds of fibrous materialssuch as filter paper, cellulose powder or the like. As a source ofnitrogen, used can be ammonium salt such as ammonium sulfate, ammoniumnitrate, ammonium acetate or the like; nitrate; and organic substancesuch as peptone, meat extract, corn steep liquor, corn gluten meal,cotton seed oil, defatted soybean or the like. Furthermore, a smallamount of inorganic metals, vitamins, growth factors, for example yeastextract containing thiamine and biotin, may be added. These mediumingredients are available as long as each concentration of them does notinhibit the growth of microorganisms. It is appropriate that a source ofcarbon is used generally at 0.025% to 0.5% by weight. It is appropriatethat a source of nitrogen is used at 0.05% to 1% by weight. Medium isgenerally adjusted to pH 6.5 to 8.5, and the resulting medium issterilized to be used. A temperature range of cultivation is availableas long as microorganisms can grow, and it can be appropriatelydetermined by a person skilled in the art. In the case of aerobicmicroorganism, for example, liquid cultivation can be performed bygeneral shake culture or airation cultivation using medium describedabove. In the case of anaerobic bacteria, liquid cultivation isperformed by a general method, for example, which comprises adding aproper concentration of reductant to medium described above to keep alow oxidation-reduction potential, and then shaking culture with theresulting medium at 20° C. to 40° C. for 24 hours to a week. As areductant, 0.03% to 0.05% of L-cysteine hydrochloride, 0.01% to 0.2% ofsodium thioglycolate, 0.001% of sodium formaldehydesulfoxylate or 0.1%of ascorbic acid can be used. To obtain enzymes, cultivation isperformed until the thing used as a source of carbon for medium such asbroken filter paper, for example, disappears.

[0011] After cultivation, a supernatant of the culture is obtained, or acrude enzyme solution is obtained by centrifuging or filtering thesupernatant of the culture. Then, the supernatant or the crude enzymesolution can be used as an agent for decomposing plant fibrous materialsof the present invention. Alternatively, the supernatant may beconcentrated by ultrafiltration, or it may be pulverized to the powderof the crude enzymes by a general method such as salting out withammonium sulfate, solvent precipitaion, dialysis or freeze-dryingmethod. Also, as an agent for decomposing plant fibrous materials, usedcan be a purified enzyme solution obtained by adding cellulose such asavicel to a crude enzyme solution, allowing a fraction showing cellulaseactivity or the like to be adsorbed thereto, and then eluting theadsorbed ingredients.

[0012] The term “vegetable waste materials” as used herein refers towaste materials generated during rice growing or a wheat production, orin producing grain such as corn and sugarcane, or in producing legumesor potatoes, and contains plant fibrous materials. Specifically,examples of vegetable waste materials are rice straw, chaff, straw,bagasse (strained sugarcane lees, core of corn) and vine of potato, andfallen leaves and chips of wood.

[0013] “A process for treating of vegetable waste materials” used hereinis not limited as long as the above agent for decomposing plant fibrousmaterials is employed. For example, the process can be performed outsideby scattering an agent for decomposing plant fibrous materials directlyover vegetable waste materials left in the field. From the viewpointthat an agent for decomposing plant fibrous materials is allowed to actefficiently, it is preferable to crush vegetable waste materials intopieces before the agent is allowed to act on it. As a way to crushvegetable waste materials into pieces, available is, for example, theway wherein rice straw or the like is harvested and cut with a combine,the way wherein tree is cut with a chain saw, the way wherein a crushingequipment to break a tree into chips is used, or the way whereinvegetable waste materials are cut or smashed with an equipment having asmashing tool or a shearing blade. In addition, it is preferable thattreatment of vegetable waste materials is performed under thetemperature condition of 20° C. to 50° C. It is preferable that when anagent for decomposing plant fibrous materials of the present inventionis used in a greenhouse wherein greenhouse cultivation or the like iscarried out, the room temperature is raised to 40° C. to 50° C. In ageneral field which exists outside, an agent to be plowed in soil,manure, fertilizer, compost and/or a soil conditioner such as abacterial preparation available on the market, a lime-nitrogen agent orthe like may be used at the same time.

[0014] The present invention is described and explained by means of thefollowing Examples, but it is to be understood that the invention is notlimited to only these Examples.

EXAMPLE 1

[0015] Fifty milliliters of L-Broth (5 g/l yeast extract, 10 g/lpeptone, 5 g/l sodium chloride) was used for pre-cultivation ofCellulomonas sp. K32A. A single colony of K32A was inoculated into thepre-culture medium, and then the pre-cultivation was carried out byshake culture (250 rpm, the amplitude of 10 mm) at 37° C. overnight in agyratory shaker (trade name: G10 Gyrotory shaker, made by New Brunswickscientific) to obtain the resulting pre-culture solution. Then, 250 mlof 1/5 PTY culture medium (1 g/l yeast extract, 1 g/l tryptone, 1 g/lpeptone) added avicel (trade mark) at 0.05% was used as a culturemedium, and the pre-culture solution was inoculated at 1% into theculture medium, and then cultivation was carried out by shake culture(250 rpm, the amplitude of 10 mm) at 37° C. until the substrate, i.e.avicel, disappeared (about 72 hours). After the cultivation, theresulting supernatant of the culture was obtained by centrifugation(6000 rpm, 10 minutes). The supernatant was then sterilized by filteringit, and proteins in the supernatant were precipitated by adding ammoniumsulfate to saturation (80%). The precipitated proteins were dissolvedand dialyzed in the buffer containing 50 mM morpholinopropanesulfonicacid (hereinafter referred to as “MOPS”), 10 mM CaCl₂ and 1 mM NaN₃. Theresultant was used as a crude enzyme solution hereinafter. The proteinconcentration in the prepared crude enzyme solution was 2 mg/ml.

[0016] To 5 ml of the crude enzyme solution, 1 g of rice straw or chaffwas added as a substrate, the mixtures were allowed to react at atemperature shown in both Table 1 and Table 2, for 72 hours or a week,respectively. Centrifugation of each resultant (3500 rpm, 2000×g, 15minutes) was carried out after the reaction, the resulting precipitationwas washed three times by adding sterilized water to it and centrifugingthe resultant in a centrifuge (a trade name: KN-70, made by KUBOTACORPORATION) (3500 rpm, 15 minutes), and then the weight of theresulting precipitation was measured in a balance after freeze-dryingit. Removal ratio was a percentage that a weight obtained by deducting aweight of resultant from the initial weight accounts for, based on theinitial weight. The results were shown in Table 1 and Table 2. TABLE 120° C. 30° C. 40° C. 50° C. Decomposed ratio of rice straw 5.7 8.0 12.218.1 after 72-hour reaction (%) Decomposed ratio of rice straw 9.8 14.522.1 32.1 after one-week reaction (%)

[0017] TABLE 2 20° C. 30° C. 40° C. 50° C. Decomposed ratio of chaff 3.55.8 7.6 11.3 after 72-hour reaction (%) Decomposed ratio of chaff afterone-week reaction (%) 9.7 13.8 21.8 18.2

EXAMPLE 2

[0018] Chaff was added at 1% as a substrate to 50 mM MOPS buffer (pH7.0) containing 10 mM CaCl₂ and 1 mM NaN₃ to prepare 50 ml of themixture in total. To the mixture was added the crude enzyme solution ofK32A obtained in EXAMPLE 1 which contained 1 mg of the proteins, andthen the resultant was incubated at 50° C. for 24 hours. Centrifugationof the resultant was carried out after the incubation, the precipitatingfraction was freeze-dried, and then the dry weight of the resultant wascompared with that of the control group (no crude enzyme solution wasadded). As a result, each weight of the resultants treated with thecrude enzyme solution was less than those of the control group by 30% to32%. In addition, when the mixture was observed before centrifugation,it is observed visually that part of chaff was dissolved.

EXAMPLE 3

[0019] Two hundred fifty milliliters of 1/5 PTY culture medium addedavicel at 0.1% which was treated with phosphoric acid was prepared underanaerobic condition, a single colony of Clostridium cellulovorans (ATCC35296) was inoculated thereto, and a cultivation was carried out byshake culture at 37° C. for 72 hours (250 rpm, the amplitude of 10 mm).After the cultivation, the resulting supernatant of the culture wasobtained by centrifugation (6000 rpm, 10 minutes). The supernatant wasthen sterilized by filtering it, and proteins in the supernatant wereprecipitated by adding ammonium sulfate to saturation (80%). Theprecipitated proteins were dissolved and dialyzed in 50 mM phosphate—12mM citrate buffer. The resultant was used as a crude enzyme solutionhereinafter. The protein concentration in the crude enzyme solution wasadjusted to 2 mg/ml.

[0020] To 5 ml of the crude enzyme solution 1 g of rice straw or chaffwas added as a substrate, the mixtures were allowed to react at atemperature shown in both Table 3 and Table 4, for 72 hours or a week,respectively. Centrifugation of each resultant (3500 rpm, 2000×g, 15minutes) was carried out after the reaction, the resulting precipitationwas washed three times by adding sterilized water to it and centrifugingthe resultant in a centrifuge (a trade name: KN-70, made by KUBOTACORPORATION) (3500 rpm, 15 minutes), and then the weight of theresulting precipitation was measured in a balance after freeze-dryingit. Removal ratio was a percentage that a weight obtained by deducting aweight of resultant from the initial weight accounts for, based on theinitial weight. The results were shown in Table 3 and Table 4. TABLE 320° C. 30° C. 40° C. 50° C. Decomposed ratio of rice straw 1.1 3.2 7.810.5 after 72-hour reaction (%) Decomposed ratio of rice straw 2.6 8.210.5 15.6 after one-week reaction (%)

[0021] TABLE 4 20° C. 30° C. 40° C. 50° C. Decomposed ratio of chaff 0.11.2 2.8 6.5 after 72-hour reaction (%) Decomposed ratio of chaff 1.2 2.05.6 8.8 after one-week reaction (%)

INDUSTRIAL APPLICABILITY

[0022] The present invention provides a beneficial process for treatingvegetable waste materials which needs less labor than labor required inprevious processes. According to the present invention, vegetable wastematerials can be allowed to be treated outside, for example, on thefields. According to the present invention, solved are problems such asthe problem of treatment costs, the problem of energy, an environmentalproblem and the problem of time for treatment which are difficult to besolved by prior art, and it is possible to treat vegetable wastematerials without depending on living conditions of microorganisms.

1. An agent for decomposing plant fibrous materials, which containsplant fibrous materials decomposing enzymes derived from amicroorganism, but not the microorganism.
 2. An agent according to claim1, wherein said microorganism belongs to Cellulomonas.
 3. An agentaccording to claim 1, wherein said microorganism belongs to Clostridium.4. A process for treating vegetable waste materials comprising allowingan agent for decomposing plant fibrous materials to act on a vegetablewaste material, said agent containing plant fibrous materialsdecomposing enzymes derived from a microorganism, but not themicroorganism.
 5. A process according to claim 4, wherein said vegetablewaste material is a member selected from the group consisting of ricestraw, chaff, straw, bagasse, vine of potato, fallen leaves and chips ofwood.
 6. A process according to claim 4, wherein said vegetable wastematerial is crushed into pieces before the agent is allowed to act onit.
 7. A process according to claim 4 which is performed under thetemperature condition of 20° C. to 50° C.
 8. A process according toclaim 4 which is performed in the fields.